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Cross-tissue transcriptomic analysis of human secondary lymphoid organ residing ILC3 reveals a default quiescent state in the absence of inflammation

ABSTRACT: A cross tissue transcriptional comparison of human ILC3 isolated from non-inflamed lymph nodes (LN) and spleens, inflamed tonsils and the peripheral blood (PB). Using uniform gating and high-purity cell-sorting, ILC3 were sorted as Lineage(CD3,CD19,CD34,CD14,CD94)-CD45intCD127+CRTH2-CD117+. From LN, spleen, tonsils and PB, NKp44- ILC3 were isolated and, in addition, NKp44+ ILC3 were sorted from spleen and tonsils. RNA quality was measured on a RNA 6000 Pico Kit (Agilent Technologies) using a 2100 Bioanalyzer (Agilent Technologies). 100 pg high-quality RNA was isolated and cDNA was generated using the SMART-Seq (v3) Ultra Low Input RNA Kit (Clontech) with 15 cycles of amplification. Amplified and Covaris-fragmented cDNA was quality-checked on a High Sensitivity DNA chip (Agilent Technologies) and further processed according the TruSeq Nano DNA Library Preparation Kit (Illumina). Data was processed as follows: SMARTer adapters were trimmed with cutadapt and the resulting sequences were aligned to the human RefSeq transcriptome using 14 TopHat2 (Kim et al., 2013). Normalization and quantification was performed using Cufflinks (Trapnell et al., 2012). FPKM counts were determined per gene with HTSeq-count.

PROVIDER: EGAS00001002636 | EGA |

REPOSITORIES: EGA

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Cross-tissue transcriptomic analysis of human secondary lymphoid organ residing ILC3 reveals a default quiescent state in the absence of inflammation

Project description:A cross tissue transcriptional comparison of human ILC3 isolated from non-inflamed lymph nodes (LN) and spleens, inflamed tonsils and the peripheral blood (PB). Using uniform gating and high-purity cell-sorting, ILC3 were sorted as Lineage(CD3,CD19,CD34,CD14,CD94)-CD45intCD127+CRTH2-CD117+. From LN, spleen, tonsils and PB, NKp44- ILC3 were isolated and, in addition, NKp44+ ILC3 were sorted from spleen and tonsils. RNA quality was measured on a RNA 6000 Pico Kit (Agilent Technologies) using a 2100 Bioanalyzer (Agilent Technologies). 100 pg high-quality RNA was isolated and cDNA was generated using the SMART-Seq (v3) Ultra Low Input RNA Kit (Clontech) with 15 cycles of amplification. Amplified and Covaris-fragmented cDNA was quality-checked on a High Sensitivity DNA chip (Agilent Technologies) and further processed according the TruSeq Nano DNA Library Preparation Kit (Illumina). Data was processed as follows: SMARTer adapters were trimmed with cutadapt and the resulting sequences were aligned to the human RefSeq transcriptome using 14 TopHat2 (Kim et al., 2013). Normalization and quantification was performed using Cufflinks (Trapnell et al., 2012). FPKM counts were determined per gene with HTSeq-count. Raw data has been deposited at the European Genome-Phenome Archive (www.ebi.ac.uk/ega) under accession EGAS00001002636.

2017-07-31 | E-MTAB-5909 | biostudies-arrayexpress

ena-DATASET-ErasmusMC-hema-04-09-2017-16:01:04:637-36 - samples

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Project description:The tumor microenvironment (TME) and somatic mutations drive progression of chronic lymphocytic leukemia (CLL). Integrated bulk and single-cell RNA sequencing (RNA-seq) of paired peripheral blood (PB) and lymph node (LN) samples from patients with treatment naïve CLL showed upregulation of oncogenic processes in LN attributable to a minor population of activated tumor cells. A 10-gene activated tumor signature was positively correlated with activated CD4+ T cells and M2 macrophages in the TME. Whole exome sequencing of paired PB and LN samples showed subclonal expansion in LN in some patients. In remaining patients with clonal stability between PB and LN, a T-cell inflamed TME was detected by RNA-seq. These data connect the TME with molecular events in the pathogenesis of CLL.

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Interdependencies of Intratumoral Genetic Heterogeneity and the Immune Microenvironment in Chronic Lymphocytic Leukemia (Bulk RNA)

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Affymetrix SNP array data for 29 MCL cases

Project description:DNA from four 29 cases, 38 tumor samples (23 PB, 12 LN, 1 Tonsil, 1 colonic biopsy, 1 spleen) and 29 normal DNA from the same patients were analyzed with Affymetrix SNP 6.0 platform for copy number alterations study. Affymetrix SNP arrays were performed according to the manufacturer's directions on DNA extracted from blood samples, lymph nodes and other tissues like spleen, tonsil and colonic biopsy

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Activation of Hydroxy-carboxylic Acid Receptor 1 effect on cardiomyocytes

Project description:Our aim is to explore the effect of Hydroxy-carboxylic Acid Receptor 1 on cardiomyocytes. Neonatal rat cardiomyocytes (NRCMs) were isolated and cultured. Subsequently, NRCMs were treated with the agonist of Hydroxy-carboxylic Acid Receptor 1 (HCAR1), 3Cl-HBA at 40 μM for 48 h(HBA1,HBA2 and HBA3) or DMSO as control(C1,C3 and C3). RNA was extracted using the KAPA RiboErase RNA-Seq kit (Roche, Basel, Switzerland), and was analysed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) for quality control. The libraries were sequenced on an Illumina HiSeq X Ten platform. DESeq2 was used to analyse RNA-seq data. Neonatal rat cardiomyocytes (NRCMs) were isolated and cultured. Subsequently, NRCMs were treated with the agonist of Hydroxy-carboxylic Acid Receptor 1 (HCAR1), 3Cl-HBA (40 μM for 48 h) or DMSO as control. RNA was extracted using the KAPA RiboErase RNA-Seq kit (Roche, Basel, Switzerland), and was analysed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) for quality control. The libraries were sequenced on an Illumina HiSeq X Ten platform.

2024-02-14 | GSE224822 | GEO

single cell transcriptomics and B-cell repertoire data from one vaccinated individual after Boostrix vaccination

Project description:The transcriptomics and BCR-rep data was meassured at the single-cell level from the longitudinal samples obtained from an individual following Boostrix vaccination. The donor had no known or suspected exposure to pertussis infection nor vaccination in the past 10 years. EDTA-blood samples for single-cell sequencing were collected at baseline and days 5 (d5), d7, d10 and d14 post-vaccination. At baseline, first 10,000 total CD19+ B cells were sorted, and after adjusting the sorting gate to exclusively sort PB/PCs, another 1,201 CD19+CD38++CD24+CD27+ PB/PCs were sorted. For subsequent visits, all available PB/PCs were sorted (11,051 cells). In total, we sorted 22,252 cells. Nearly 22,000 B cells enriched in PB/PCs from different time-points were processed into single cells in a Chromium Controller (10X Genomics). Reverse transcription PCR and library preparation were carried out under the Chromium Single Cell 3’ v3 protocol (10X Genomics) per manufacturer’s recommendations. After amplification of the cDNA, a 5 ́gene expression library and paired heavy and light chain library were generated from cDNA of the same cell using Chromium Single Cell VDJ reagent kit (scBCR-rep library; v1.1chemistry, 10X Genomics). After library preparation, quality control was performed using a bioanalyzer (Agilent 2100 Bioanalyzer; Agilent Technologies). The libraries were sequenced in the NovaSeq6000 sequencer (Illumina) using the v1.0 sequencing reagent kit (read length: 2 x 150bp).

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